We are indebted to Alice Gaughan and Jiao-Jing Wang whose outstanding technical support, intellectual input, and moral support were instrumental in moving these studies ahead. These studies were supported by the NIH grant AI036532 to GAH.

1. Lethal Irradiation
<ol>
 <li>Place up to 10 recipient mice into a microisolator cage compatible with the irradiator to be used.</li>
 <li>Irradiate in 2 equal doses summing total dose (total dose = 9 cGy for BALB.B recipients). Second irradiation should be 3 hr after first. Injection should occur between 4-6 hr after final irradiation. Irradiate mice in either Cs137 source or RS2,000, irradiator.</li>
 <li>Following the second radiation dose, store mice in microisolator cage with acidified water until the time of transplant.</li>
</ol>
2. Splenocyte Preparation
<ol>
 <li>Euthanize one donor mouse according to institutional guidelines. Each donor generally yields 75x106 - 125x106 splenocytes. Using CD8 purification kits, each mouse generally yields 6x106 - 12x106 CD8 T cells.</li>
 <li>Remove the spleen by first making a vertical 2 cm incision 2 cm right of the midline directly under the rib cage. Cut through the fur, skin, and visceral membrane.</li>
 <li>Remove spleen and create single cell suspension by placing spleen in 40 &mu;l mesh screen in a Petri dish with 1640 RPMI with 5% FBS. Use a syringe plunger to break apart the spleen until the entire spleen has been passed through mesh screen.</li>
 <li>Repeat this process for each WT and L2G85.B6 donor. Collect all the WT single cell suspensions in 1-50 ml conical tube, and collect all the L2G85.B6 single cell suspensions in a separate 50 ml conical tube.</li>
 <li>Centrifuge cells at 1,200 rpm for 10 min at 4 &deg;C. Resuspend pellets in 1640 RPMI with 5% FBS and count cells.</li>
</ol>
3. Bone Marrow Preparation
<ol>
 <li>Remove skin from one hind limb of a donor mouse.</li>
 <li>Carefully cut away as much muscle tissue as possible from femur and tibia/fibula.</li>
 <li>Remove hind limb by cutting femur away at the hip joint. Cut away rear paw just below tibia/fibula intersection. All bone cuts should be made using sturdy scissors.</li>
 <li>Carefully remove any remaining muscle tissue. Cut off fibula-relatively little bone marrow is found in fibula and not worth the effort.</li>
 <li>Place hind limb in cold 1640 RPMI 5% FBS and repeat process with the second hind limb. Each mouse should yield between 20x106 - 40x106 bone marrow cells.</li>
</ol>
4. Bone Marrow Removal
<ol>
 <li>Remove one hind limb from media and place in large Petri dish with a small amount of cold media (~1 ml).</li>
 <li>Cut away the knee joint. Using a syringe (volume &gt; 5 ml), insert subcutaneous needle into tibia and depress syringe until all red material is removed from interior of tibia. Repeat process with femur. Discard remaining bones devoid of bone marrow. Repeat process with second hind limb.</li>
 <li>Create single cell suspension by pipetting media containing bone marrow into large Petri dish and use syringe plunger and 40 &mu;l mesh screen. Pipette single cell suspension into 50 ml conical tube and keep on ice.</li>
</ol>
5. CD3 Depletion
There are a variety of ways to deplete CD3+ cells from the bone marrow. Our lab uses a kit made by Miltenyi Biotec (CD3-biotin - 130-093-021). Deplete CD3+ cells from the bone marrow following manufacturer's protocol. The buffer for Miltenyi kits will henceforth be called MACS Buffer (2 mM EDTA, 0.5% BSA in PBS, pH 7.2).
<ol>
 <li>Wash cells by centrifuging splenocytes and bone marrow cells at 1200 rpm for 10 min at 4 &deg;C and count cells.</li>
 <li>Remove ALL supernatant. Resuspend bone marrow cells in 100 &mu;l per 10 million bone marrow cells in MACS Buffer.</li>
 <li>Continue with CD3 depletion using manufactures' protocol.</li>
 <li>Wash CD3 depleted bone marrow cells 3 times in sterile PBS. Count CD3 depleted bone marrow cells and resuspend at an appropriate volume to inject 107 cells.</li>
</ol>
6. L2G85.B6 CD8+ T Cell Purification
There are a variety of ways to purify CD8 T cells from L2G85.B6 mice. Also, there are several ways to deplete CD8 T cells from WT splenocyte donors. Our lab uses kits from Miltenyi Biotec (CD8 depletion - 130-049-401, CD8 purification - 130-095-236).
<ol>
 <li>Resuspend the WT pellet in 90 &mu;l MACS Buffer per 107 cells. Continue with CD8 T cell depletion according to manufactures' protocol (CD8 depletion - 130-049-401).</li>
 <li>Resuspend the L2G85.B6 pellet in 40 &mu;l MACS Buffer per 107 cells. Continue with CD8 T cell purification according to manufactures' protocol (CD8 purification - 130-095-236).</li>
 <li>Count each cell population and wash each population 3 times with sterile PBS. Resuspend each population in appropriate volume to inject 18x106 WT (CD8 depleted) splenocytes and 2x106 L2G85.B6 purified CD8 T cells.</li>
</ol>
7. Injection Preparation
<ol>
 <li>Combine 107 CD3 depleted bone marrow cells, 18x106 WT (CD8 depleted) splenocytes, and 2x106 L2G85.B6 purified CD8 T cells in a microcentrifuge tube. Wash with sterile PBS and resuspend in 300 &mu;l.</li>
 <li>Inject cell preparation into the tail vein of recipient mice. Injections should be done using 28 gauge needles.</li>
 <li>Store mice in microisolator cage with acidified water. Score mice daily using scoring GVHD scoring system developed by Cooke et al1.</li>
</ol>
8. Bioluminescent Imaging
<ol>
 <li>Six days post transplant, inject recipient mice with 4 mg D-luciferin. Allow 5 min for luciferin to react with luciferase.</li>
 <li>Anesthetize mouse in isoflurane chamber of bioluminescence imager and image recipients for 5 min with small binning. This will create a high resolution image while gathering as many events as possible.</li>
 <li>Analyze data using Living Image software. The scale of the pseudo-color image can be changed to yield the best results. However, it is imperative that the same scale be used across experiments.</li>
 <li>Regions of interest can be created using Living Image software and light emittance can be quantified by calculating the flux (photons/sec) being emitted from each region of interest.</li>
</ol>
9. Representative Results
Approximately 7-10 days post transplant, mice begin showing clinical signs of GVHD. Mice appear scruffy due to lack of grooming. Recipients will also begin to lose weight between 7-10 days post transplant. Activity and posture of recipient mice will remain relatively normal until approximately day 12-14 post transplant. Cumulative GVHD scores will steadily increase through the first 2-3 weeks post transplant (Figure 1A). Disease course is quite variable between mice; however, recipients should uniformly succumb to GVHD by 30-40 days post transplant (Figure 1B).
Figure 2 shows recipient mice that were imaged 6 days post transplant. Pseudo-colored scale shows varying light emittance throughout the body with the highest light intensity being emitted in the spleen and the gut. CD8 T cell accumulation in the gut is consistent with previous findings6. Recipient mice can be placed back in microisolator cage to be imaged at a later time point or euthanized for ex vivo imaging.
<table border="1">
 <tbody>
 <tr>
 <td>Criteria</td>
 <td>Grade 0</td>
 <td>Grade 1</td>
 <td>Grade 2</td>
 </tr>
 <tr>
 <td>Weight loss (wkly.)</td>
 <td>&lt; 10%</td>
 <td>&gt; 10% - &lt; 25%</td>
 <td>&gt;25%</td>
 </tr>
 <tr>
 <td>Posture</td>
 <td>Normal</td>
 <td>Hunching only at rest</td>
 <td>Several hunching; impairs movement</td>
 </tr>
 <tr>
 <td>Activity</td>
 <td>Normal</td>
 <td>Mild to moderate decrease in activity</td>
 <td>Stationary unless stimulated</td>
 </tr>
 <tr>
 <td>Fur texture</td>
 <td>Normal</td>
 <td>Mild to moderate ruffling</td>
 <td>Severe ruffling/poor grooming</td>
 </tr>
 <tr>
 <td>Skin texture</td>
 <td>Normal</td>
 <td>Scaling of paws/tail</td>
 <td>Obvious areas of denuded skin</td>
 </tr>
 </tbody>
</table>
Table 1. Cooke et al developed this scoring system in 19961. Mice should be scored daily on each of the criteria on the left. Each mouse is given a score of 0-2 for each criteria and the total score is a sum of all the individual scores.
<img alt="Figure 1" src="/files/ftp_upload/3697/3697fig1.jpg" /> 
Figure 1. Lethally irradiated BALB.B were transplanted with 107 bone marrow cells alone or with 18x106 CD8 T cell depleted WT splenocytes and 2x106 purified L2G85.B6 CD8 T cells. A) Clinical score data of recipients of bone marrow alone or with CD8 T cell depleted WT splenocytes and purified L2G85.B6 CD8 T cells. B) Survival data of recipients of bone marrow alone or with CD8 T cell depleted WT splenocytes and purified L2G85.B6 CD8 T cells. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/3697/3697fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3697/3697fig2.jpg" /> 
Figure 2. Lethally irradiated BALB.B mice were transplanted with 107 bone marrow cells alone or with 18x106 CD8 T cell depleted WT splenocytes and 2x106 purified L2G85.B6 CD8 T cells. Recipients were injected with 4 mg D-luciferin via intraperitoneal injection and were imaged using Xenogen IVIS for 5 min at small binning. Pseudo-colored images are shown where purple represents low intensity and red represents high intensity Regions of interest were drawn around the entire mouse and total flux (photons/sec) were quantified.
<img alt="Figure 3" src="/files/ftp_upload/3697/3697fig3.jpg" /> 
Figure 3. Lethally irradiated BALB.B mice were transplanted with 107 bone marrow cells alone or with 18x106 CD8 T cell depleted WT splenocytes and 2x106 purified L2G85.B6 CD8 T cells. Recipients were injected with 4 mg D-luciferin via intraperitoneal injection and were imaged using Xenogen IVIS for 5 min at small binning. Pseudo-colored images are shown where purple represents low intensity and red represents high intensity. Recipients were imaged on A) day 4, B) day 6, and C) day 8 post transfer.

<ol>
	<li>Cooke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+of+idiopathic+pneumonia+syndrome+after+bone+marrow+transplantation:+The+roles+of+minor+H+antigens+and+endotoxin.">An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: The roles of minor H antigens and endotoxin.</a> Blood. 88, 3230-3239 (1996).</li><li>Berger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+subsets+involved+in+lethal+graft-versus-host+disease+directed+to+immunodominant+minor+histocompatibility+antigens.">T cell subsets involved in lethal graft-versus-host disease directed to immunodominant minor histocompatibility antigens.</a> Transplantation. 57, 1095-1102 (1994).</li><li>Nimer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+depletion+of+CD8++cells+for+prevention+of+graft-versus-host+disease+after+bone+marrowtransplantation.+A+randomized+controlled+trial.">Selective depletion of CD8+ cells for prevention of graft-versus-host disease after bone marrowtransplantation. A randomized controlled trial.</a> Transplantation. 57, 82-87 (1994).</li><li>Korngold, R., Sprent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+markers+of+T+cells+causing+lethal+graft-vs-host+disease+to+class+I+vs+class+II+H-2+differences.">Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences.</a> Journal of Immunology. 135, 3004-3010 (1985).</li><li>Cao, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+using+labeled+donor+tissues+reveals+patterns+of+engraftment,+rejection,+and+survival+in+transplantation.">Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation.</a> Transplantation. 80, 134-139 (2005).</li><li>Asady, R. E. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-{beta}-dependent+CD103+expression+by+CD8(+)+T+cells+promotes+selective+destruction+of+the+host+intestinal+epithelium+during+graft-versus-host+disease.">TGF-{beta}-dependent CD103 expression by CD8(+) T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease.</a> J. Exp. Med. 201, 1647-1657 (2005).</li><li>Larson, R. S., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+leukocyte+integrins.">Structure and function of leukocyte integrins.</a> Immunol Rev. 114, 181-217 (1990).</li><li>Karecla, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+E-cadherin+on+epithelial+cells+by+the+mucosal+T+cell+integrin+alpha+M290+beta+7+(alpha+E+beta+7).">Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin alpha M290 beta 7 (alpha E beta 7).</a> Eur. J. Immunol. 25, 852-856 (1995).</li><li>Cepek, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+between+epithelial+cells+and+T+lymphocytes+mediated+by+E-cadherin+and+the+alpha+E+beta+7+integrin.">Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin.</a> Nature. 372, 190-193 (1994).</li><li>Malarkannan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+functional+characterization+of+a+dominant+minor+H+antigen,+H60.">The molecular and functional characterization of a dominant minor H antigen, H60.</a> J. Immunol. 161, 3501-3509 (1998).</li></ol>

No conflicts of interest declared.

The protocol for inducing GVHD in mice presented here represents a clinically relevant model of murine GVHD. Originally established by Berger et al. in 1994, the C57Bl/6 into BALB.B strain combination is MHC-matched, with GVHD mortality mediated by CD4 dependent, CD8 T effectors2, highly similar to the most common clinical scenario3. It is known that transplanting CD8 T cells alone does not cause GVHD in this model; however, disease progression is significantly worse when both CD4 and CD8 T...

<table><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>12633-012</td><td></td></tr><tr><td>Fetal Calf Serum</td><td>Invitrogen</td><td>10439016</td><td></td></tr><tr><td>40 &amp;mu;M Cell Strainer</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>CD3e-Biotin </td><td>Miltenyi Biotec</td><td>130-093-021</td><td></td></tr><tr><td>Anti-Biotin Microbeads</td><td>Miltenyi Biotec</td><td>130-091-147</td><td></td></tr><tr><td>CD8a Microbeads</td><td>Miltenyi Biotec</td><td>130-049-401</td><td>Used to deplete CD8 T cells from spleen.</td></tr><tr><td>CD8a Purification Antibody Cocktail</td><td>Miltenyi Biotec</td><td>130-095-236</td><td>Used to purify CD8 T cells from spleen.</td></tr><tr><td>D-Luciferin</td><td>Caliper Life Sciences</td><td>122796</td><td></td></tr></tbody></table>

induction of graft-versus-host disease and in vivo t cell monitoring using an mhc-matched murine model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Watch this Scientific Journal Video about Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model at JoVE.com

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

induction-graft-versus-host-disease-vivo-t-cell-monitoring-using-an

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.6K Views.  The Ohio State University Medical Center. Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Video: Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Cancer Research

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Medicine

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Summary

Explore More Videos

Chapters in this video

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8⁺ T Cells in Mice

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease